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Volume 1, Issue 2, March – April 2010; Article 021

ISSN 0976 – 044X

DETERMINATION OF ESTRADIOL VALERATE IN PHARMACEUTICAL
PREPARATIONS BY ZERO - AND FIRST-ORDER DERIVATIVE
SPECTROPHOTOMETRIC METHODS
B. YILMAZ
Department of Analytical Chemistry, Faculty of Pharmacy, Ataturk University, 25240, Erzurum, Turkey
E-mail: bilalylmaz@yahoo.com

ABSTRACT
This paper describes zero- and first-order derivative spectrophotometric methods for determination of estradiol valerate in
pharmaceutical preparations. The solutions of the standard and pharmaceutical samples were prepared in methanol. Absorbances of
estradiol valerate were measured at 280 nm for zero-order by measuring height of peak from zero, at 292 nm for first-order derivative
spectrophotometric method by measuring peak to peak height. The linearity ranges were found to be 1.25-12.5 µg mL-1 for the zero and
first-order derivative spectrophotometric methods. The developed methods in this study are accurate, sensitive, precise, and reproducible
and can be directly and easily applied to the pharmaceutical preparation. Also, the results obtained from two spectrophotometric methods
were compared and no significant difference was found statistically.
Keywords: Estradiol valerate, Zero-, First-order derivative spectrophotometric method, Pharmaceutical preparation.

INTRODUCTION
Estradiol valerate (Figure 1) is a synthetic estrogen that is
very significant in clinical medicine. Estradiol valerate can
be used to treat menopause syndrome and prostate cancer,
and can be used together with progestogen for the
inhibition of ovulation [1].

spectrophotometric methods were also carried out and all
optimization parameters were also considered. Also, the
developed methods were applied to commercial
preparation as dragee.
MATERIALS AND METHODS
Chemicals and reagents
Estradiol valerate was obtained from Sigma-Aldrich (St.
Louis, MO, USA). Climen dragee containing 2 mg
estradiol valerate (Schering Pharmaceutical Industry,
Istanbul, Turkey) was used for analysis.
Instrumentation

Figure 1: Chemical structure of estradiol valerate
Several analytical methods have been reported for
determination of estradiol valerate including colorimetry
[2], fluorimetry [3, 4], gas chromatography [5] gas
chromatography-mass spectrometry (GC-MS) [6] and high
performance liquid chromatography (HPLC) [7, 8].
To our knowledge, there is no derivative spectroscopic
method for determination of estradiol valerate in
pharmaceutical preparation in literature. Derivative
spectrophotometry is an analytical technique of great
utility for extracting both qualitative and quantitative
information from spectra composed of unresolved bands,
and for eliminating the effect of baseline shifts and
baseline tilts. It consists of calculating and plotting one of
the mathematical derivatives of a spectral curve [9]. In the
last year, this technique has been rapidly gained its
application in the analysis of pharmaceutical preparations.
We wanted to develop new spectrophotmetric methods for
determination of estradiol valerate in pharmaceutical
preparation without the necessity of sample pre-treatment.
After developing zero- and first-order derivative

A
Thermospectronic
double-beam
UV-Visible
spectrophotometer (HEIOS) with the local control
software was used. Zero- and first-order derivative spectra
of reference and sample solutions were recorded in 1 cm
quartz cells at a scan speed of 600 nm min-1, a scan range
of 250-310 nm and fixed slit width of 2 nm.
Preparations of the standard and quality control
solutions
The stock standard solution of estradiol valerate was
prepared in methanol to a concentration of 50 g mL-1 and
kept stored at -20 0C. Working standard solutions were
prepared from the stock standard solutions. A calibration
graph was constructed in the range of 1.25, 2.5, 5, 7.5, 10
and 12.5 g mL-1 for estradiol valerate (n=6). For quality
control samples containing 2, 7, 11 g mL-1 of estradiol
valerate, the stock solution was diluted with methanol.
Procedure for pharmaceutical preparation
Climen dragee drug was weighed and finely powdered.
The average weight of drug was determined with the help
of weight of 10 dragees. A portion of powder equivalent to
the weight of one dragee was accurately weighed into
100 mL volumetric flask and 70 mL methanol was added.
The volumetric flask was sonicated for 15 min to effect

International Journal of Pharmaceutical Sciences Review and Research
Available online at www.globalresearchonline.net

Page 112

the noise decreases by increasing Δλ.5 and 10 g mL-1) International Journal of Pharmaceutical Sciences Review and Research Available online at www. As demonstrated in the Figure 3. These two shouldered peaks were separated by using derivative spectrophotometer. version 10. Maximum is represented at 270 nm and minima are shown at 282 and 292 nm. The solution was filtered through a Whatman No 42 paper. Article 021 ISSN 0976 – 044X complete dissolution of the estradiol valerate.order derivative spectrophotometric method. Approximate dilutions were made at concentrations of 2. the ratio of signal/noise elevates and the sensitivity of the method increases by controlling the degree of low pass filtering or smoothing. respectively. the solution was then made up to volume with methanol. Zero.5 nm) for first.5 and 10 g mL-1 with methanol. Correlations were considered statistically significant if calculated P values were 0. Therefore. Figure 3: First-order derivative spectrum of standard solution of estradiol valerate As no difference was observed between spectra of estradiol valerate standard and dragee solutions and in the maximum wavelengths of all spectra.and first-order derivative spectra of estradiol valerate (Figures 4. Generally. March – April 2010. Figure 3 presents the overlay of first-order ultraviolet spectra of estradiol valerate standard samples in methanol.net Page 113 .5 and 10 g mL-1) Figure 2: Zero-order derivative spectrum of standard solution of estradiol valerate Figure 5: First-order derivative spectrum of solutions of Climen dragee containing estradiol valerate (2. n=5 (Δλ=17. Optimum results were obtained in the measuring wavelength range 250-310 nm.globalresearchonline. 5). Figure 4: Zero-order derivative spectrum of solutions of Climen dragee containing estradiol valerate (2. the spectra present characteristic a maximum and two minima.and first-order derivative spectra were recorded against methanol.0.05 or less. Issue 2.Volume 1. a series of n values (n=1-9) were tested in the first-order derivative spectra of estradiol valerate in methanol. Figure 2 presents the overlay of UV spectra of estradiol valerate in methanol gives two characteristic maxima at 280 and 289 nm. RESULTS AND DISCUSSION Method development and optimization The derivative wavelength difference (Δλ) depends on the measuring wavelength range and n values (smoothing factor). Data analysis All statistical calculations were performed with the Statistical Product and Service Solutions (SPSS) for Windows. it was suggested that the developed methods allowed complete elimination of the background absorption due to the capsule excipients at the chosen wavelengths both in zero. Optimal wavelength range should be chosen since the broad peaks become sharper.

3 σ/S and 10 σ/S. Method validation Linearity Limits of detection (LOD) and quantitation (LOQ) For quantitative analysis of estradiol valerate. aBased on six calibration curves. x: estradiol valerate concentration (g mL-1).S.and first-order derivative spectrophotometric methods and to study the interference of formulation additives.71 1.S.43 3. The peak to zero method for calibration curve in the first-order derivative spectrophotometric method was used. Table 1.globalresearchonline.186 2.72 2.94 1.399 1. Three different concentrations which were quality control samples (2. According to the results obtained.003 0.and first-order derivative spectrum of estradiol valerate in standard and drug formulation (Climen dragee) solutions show that the wavelength of maximum and minimum absorbance did not changed (Figures 4.D%a 1.132x+0.00% and ≤3.209 10.029 7.27 3.50 1.50 1.071 nm D292 nm =0. Sb: Standard deviation of slope of regression line.29 Precision R. aAverage of six replicate determinations.130.265 1.91 1.115 0.160.85 Precision R.and first-order derivative spectrophotometric methods.9994 0.120.956x-0.5%-99.00 1.8% (Table 3). Accuracy and precision The precision of the analytic methods were determined by repeatability (within-day) and intermediate precision (between-day).09% (n=6) and intermediate precision was ≤3. International Journal of Pharmaceutical Sciences Review and Research Available online at www.026 7.032 7. respectively (Table 2). 10 g mL-1) and analytical recovery experiments were performed by adding known amount of pure drugs to pre-analyzed samples of commercial dosage form (Climen). LR: Linear regression Sa: Standard deviation of intercept of regression line.345 Between-day 0. Repeatability was ≤2.060.25-12.81 Accuracy S. Article 021 ISSN 0976 – 044X standard error of the slope and the intercept are given in Table 1.81% (n=6) for zero.246 11 11. 6.011 0.800.D: Standard deviation of six replicate determinations.net Page 114 . LOD: Limit of detection.09 1.82 3.and first-order derivative spectrophotometric methods were between 101.040.449 Sa Sb R LOD LOQ 0.203 : Wavelength.47 FoundSD (g mL-1) 2.021 7.5 1 =0.221 10.25-12.25-12.55 3.00 3. The linearity ranges of all spectrophotometric methods were found to be 1. Accuracy of zero. the zero.D%a 1.212 0.S.71 -1.2%-101. 5).45 3.010. the recovery was checked as three different concentration levels (2. The recoveries of zero.5 µg mL-1. R: Coefficient of correlation. March – April 2010. LOQ: Limit of quantitation. the calibration curves were plotted for each spectrophotometric method over the concentration ranges cited.00% (n=6). Results of regression analysis of estradiol valerate by the proposed methods Range (g mL-1) Methods Zero-order Spectrophotometric Method First-order Spectrophotometric Method LRa A280 1.Volume 1.220.5% and 98.9995 0. respectively (Table 2). LOD and LOQ values were calculated as 3. The statistical parameters and regression equations which were calculated from the calibration curves along with the The LOD and LOQ of estradiol valerate by the proposed methods were determined using calibration standards.427 Accuracy 2.110.04 2. 1D: First-order absorbance Table 2. Issue 2.00 1.94% and ≤3.and first-order derivative spectrophotometric methods showed acceptable relative error values were ≤2.10% and ≤3.D (g mL-1) 2. where S is the slope of the calibration curve and σ is the standard deviation of y-intercept of regression equation (n=6) [10] (Table 1).5 1. The percent analytical recovery values were calculated by comparing concentration obtained from the spiked samples with actual added concentrations.10 2. 7.900. Accuracy: (%relative error) (found-added)/addedx100 Specificity Comparison of the zero.030. Recovery To determine the accuracy of the zero. methods can be considered specific. A: Absorbance. R.226 2.and first-order derivative spectrophotometric methods are able to access estradiol valerate in presence of excipients and hence. Precision and accuracy of estradiol valerate by the proposed methods  (nm) Method Zero-order Spectrophotometric Method First-order Spectrophotometric Method A280 1 nm D292 nm Added (g mL-1) Within-day 2 7 11 2 7 FoundS.090.397 1.57 -0.09 11. respectively.D: Relative standard derivation. 11 g mL-1) were analyzed six time in one day for within-day precision and once daily for three days for between-day precision.016 0.

7±2.D : Standard deviation of six replicate determinations.01 t values tc=1. +40C (Recovery %  S.87 2.5±0.D) Room temperature stability (Recovery %  S. X: mean.14 98.17 101. R. tc: Calculated F values.34 6.04 102.2 1.09 102.7±0.8 3.366 S.S. Stability of estradiol valerate in solution  (nm) A280 nm 1 D292 nm Refrigeratory stability. Recovery values of estradiol valerate in pharmaceutical preparation Commercial preparation Added Method  (nm) (g mL-1) Zero-order 2 Spectrophotometric A280 nm 6 Method 10 2 1 First-order D292 6 Spectrophotometric nm 10 Method Climen dragee (2.92 93.06 101.D) Stability (%) Added (g mL-1) 3 8 12.14 102. aAverage of six replicate determinations Table 6.94 1.62 98.D) 24 h 72 h 24 h 72 h 24 h 72 h 101.1±0.01 0.02 D292 nm 18 1.S.97 98.4±0.D: Relative standard derivation. tt: Tabulated t values.2±0.49 5.and first-order derivative spectrophotometric methods. Article 021 ISSN 0976 – 044X frozen (-200C) temperature for 24 h and 72h.54 102.24 98. Stability measurements were carried out with zero.19 101.5±0. The results were evaluated comparing these measurements with those of standards and expressed as percentage deviation and estradiol valerate was found as stable at room temperature.Volume 1.52 103. Determination of estradiol valerate in pharmaceutical preparation Commercial preparation Method Climen dragee 2 mg Zero-order Spectrophotometric Method First-order Spectrophotometric Method 1  (nm) n Found  S.0±1.1±1.3±1. 4 and -200C for at least 72h (Table 4).05) International Journal of Pharmaceutical Sciences Review and Research Available online at www.99-2.4±0.01 S.59 101.D Std.5 1.72 99.3 2.22 102.44 10.06 103.08 101. R.386 99.net Page 115 .97-2.980.D: Standard deviation.07 98.3±0.4±0.5 g mL-1) Recovery R.06 99.8±0.5±0.147 98.5±0.970.990.038 101.4±2.globalresearchonline.149 99.1±1.69 n: Number of determination.980. Statistical comparison (t-test) of the results obtained by proposed methods Commercial preparation Climen dragee 2 mg Statistical Values n X S. These solutions were stored at room temperature.2±2.2±0.8±0.3±0.09 101.13 tt=1.1±2.7 2. S.452 100.2±0.08 99.99 0.08 1.D a FoundS.0±0.5 2.7±0.68 1.01 101.6±1.D -1 (%) (%) (g mL ) 101.52 99.452 1. Stability To evaluate the stability of estradiol valerate.D: Relative standard derivation.65 101.97-2.D : Standard deviation of six replicate determinations Table 5.5 2.6±1.35 1. . Ho: Hypothesis: no statitically significant difference exists between two methods.15 107.3±1. tt tc: Ho hypothesis in accepted (=0.99-2.55 S.64 100.45 101. medium and higher ranges of calibration curve for different temperature and times.5 2.2±2.5 3 8 12.07 98.02 First-order derivative Spectrophotometric Method 18 1.Error CI Zero-order derivative Spectrophotometric Method 18 2.20°C (Recovery %  S.D (mg) Recovery (%) R.5±0.080.180.030.84 99.8±0.058 99. standard solutions were prepared separately at concentrations covering the low.4±0. a Average of six replicate determinations Table 4.67 9.386 1.87 101.21 99.D: Standard deviation of six replicate determinations.142 101.21 98. CI: Confidence interval.010.S.5±0.5 Frozen stability.Da (%) Confidence interval A280 nm 18 2.64 97.82 102.4±0. Issue 2. refrigeratory (40C) and Table 3. March – April 2010.0±4.15 1.S.

Cheng JP. 2010. Chen GN.. [3] James T. secondand third-order derivatıve spectrophotometric method. zero..D. [6] Daseleire EA.1138-1141. Biomed. The proposed methods are very effective for the assay of estradiol valerate in dragees. 30. 1986. Pharm. Pharm.and first-order derivative spectrophotometric methods were statistically compared using the t-test.Volume 1.. 674-680. J. [5] Zuleski FR.. Each level was repeated six times. The validity of the proposed methods was presented by recovery studies using the standard addition method. J. Determination of metoprolol in pharmaceutical preparations by zero-. Sci. 56. 67. Chromatogr. Acta. Wei JL. [8] Wei JK. At 95 % confidence level.net Page 116 . there is no significant difference between zeroand first-order derivative spectrophometric methods. Pharm. ************ International Journal of Pharmaceutical Sciences Review and Research Available online at www.. 161-164. Loh A. 367. Rapid. 124. CONCLUSION In conclusion. Peteghem CH. J. developed methods can be recommended for routine and quality control analysis of estradiol valerate. 1992. [4] Fishman S. Anal. J. 86-87. REFERENCES [1] Duan JP. Fluorometric determination of estradiol valerate in sesame oil or ethyl oleate injectables. 64. Di Carlo FJ. The apparatus and reagents used seem to be accessible even for the simple laboratories.13). [9] Ojeda CB. Sci. sensitive colorimetric method for determination of ethinyl estradiol. 124.D and R. The best results obtained at 280 nm and 292 nm for zero. Wub XP. 1221-1223. Article 021 ISSN 0976 – 044X Comparison of two spectrophotometric methods Zero and first-order derivative spectrophotometric methods were applied for determination of the commercial dragee (Table 5). 1990. 409-414. first-. Guesquiere A. J.. This is suggested that the two methods are equally applicable. Therefore. Chromatogr. Chen HQ. No interference from the common excipients was observed. March – April 2010. Sci. 1-15. Rojas FS. Collaborative study. Determination and stability study of oestradiol benzoate in a pharmaceutical ointment by HPLC. Chen ML. 1651-1655. 2004. Isocratic RPHPLC determination of twelve natural corticosteroids in serum with on-line ultraviolett and fluorescence detection. Nicolas A. Determination of estrogens in dosage forms by fluoroscence using dansyl Chloride. no significant difference was found between the proposed spectrophotometric methods (tt=1.and first-order derivative spectrophometric methods were developed for determination of estradiol valerate in dragee dosage form.globalresearchonline. Chim. a known amount of reference drug was spiked to formulated dragees and the nominal value of drug was estimated by the proposed methods. Issue 2.69>tc=1. 1973. Multiresidue analysisof anabolic agents in muscle tissues and urines of cattle by GC-MS. 64. Adsorptive and electrochemical behaviors of estradiol valerate at a mercury electrode. Chromatogr. Estradiol valerate can be directly determined in dragees in presence of excipients without sample pre-treatment procedures by using spectrophotometric methods.. 518. For this purpose. International Journal of Pharma and Bio Sciences. 1975. Assoc. Elshaburi SR. [10] Yilmaz B. Benoit E. 1978. 1975. 1999.S. Chem. Tawfik AS. II. Therefore. 428-433. Analyst. Zhou XT. Anal. [7] Leroy P. [2] Eldawy MA. Recent developments in derivative ultraviolet/ visible absorption spectrophotometry. Sci. The results were reproducible with low S. the calculated tvalues do not exceed the theoretical values (Table 6). Off. J. 4. Also. Determination of ethinyl estradiol in human urine by radiochemical GLC. 1. The results show the high reliability and reproducibility of two methods.